Lateral habenula glutamatergic neurons projecting to the dorsal raphe nucleus promote aggressive arousal in mice

The dorsal raphe nucleus (DRN) is known to control aggressive behavior in mice. Here, we found that glutamatergic projections from the lateral habenula (LHb) to the DRN were activated in male mice that experienced pre-exposure to a rival male mouse (“social instigation”) resulting in heightened intermale aggression. Both chemogenetic and optogenetic suppression of the LHb-DRN projection blocked heightened aggression after social instigation in male mice. In contrast, inhibition of this pathway did not affect basal levels of aggressive behavior, suggesting that the activity of the LHb-DRN projection is not necessary for the expression of species-typical aggressive behavior, but required for the increase of aggressive behavior resulting from social instigation. Anatomical analysis showed that LHb neurons synapse on non-serotonergic DRN neurons that project to the ventral tegmental area (VTA), and optogenetic activation of the DRN-VTA projection increased aggressive behaviors. Our results demonstrate that the LHb glutamatergic inputs to the DRN promote aggressive arousal induced by social instigation, which contributes to aggressive behavior by activating VTA-projecting non-serotonergic DRN neurons as one of its potential targets.

7. There are some inappropriate statements: "In particular, continuous activation of the LHb-DRN projection prior to an aggressive encounter caused a strong escalation of aggressive behaviors". This is not a proper interpretation of Figure 4 as the ON/ON regime does not distinguish if stimulation before or during aggressive encounter elevates aggression.
"Thus, our data show that depending on the subregion of the LHb or neural projection targets, activation of LHb neurons can have different effects on aggressive behavior." This manuscript does not have data on LHb subregions. 8. In Figure 6G, eArchT-EYFP does not delimit the contour of a cell. How to use YFP signals to determine if mcherry cells are YFP positive? 9. DRN neurons project to many brain regions besides VTA. Why focus on the VTA projections?
10. In the experiment of optogenetic activation of the DRN-VTA projections, an on/off session needs to be added to test if activating these project¬¬ions mimics social instigation.
11. An off-off-off session should be added to Fig. 7 to test if the decrease over time of the duration of total aggressive behaviors (Fig. 7g) and locomotion (Fig. 7j) is due to habituation effects.
Reviewer #2 (Remarks to the Author): Takahashi and colleagues investigated the role of lateral habenula (LHb) input to the dorsal raphe (DRN) in mediating aggression. Their results suggest that glutamatergic LHb input to the DRN plays a key role in escalating aggression, but not in basal aggressive behaviour. The authors went on to show that DRN neurons that receive LHb input project to the ventral tegmental area (VTA). Optogenetic activation of the latter projection (DRN-VTA) also increases aggressive behaviour.
Overall, the study is well designed and the topic is timely. The authors have used state-of-the-art techniques and as far as I can see almost all important control experiments were performed to justify the conclusion mentioned above.
I am certain that this study is of interest to a wider community, and thus, the study deserves publication in this journal.
I only have a few minor comments: -Re Figure 1 -you do give numbers for the Vglut2-positive cells ("we confirmed that 99.2% of RetroBead-positive cells were colocalized with Vglut2 (245 cells among 247 cells analyzed; Fig. 1ln)…."), but you don't give any numbers for the cFos-positive neurons in the text. Should be around 20-40 cells according to Figure 1i. Again, assuming these numbers represent all the LHb cells being activated during the task.
- Figure 2f -There is a typo: Freqency. Actually, the typo seems to be present in all figures.
-Re experiments in respect to Figure 4 -here, you stated that you waited 5 weeks for the expression of the constructs. Is there a specific reason for that? I am asking as you mentioned three weeks for the other experiments.
-In terms of the electrophysiological experiments you tested the Arch construct in vitro (Figure 5b), but for the other paradigms you did not test the constructs and basically their efficacy (e.g. for the hM4D-, NpHR-and ChR2-experiments). For example, did the one-minute light stimulation you applied to activate LHb-DRN projections ( Figure 4) activate DRN neurons throughout? In other words, does a shorter activation period do the same job? -Extended Data Figure 3 -does the finding hold true for the right and left LHb? Did you identify any functional asymmetry? Or did you only inject into either the right or left? Please indicate.
-Extended Data Fig. 4e and j -It is difficult to recognise the legend colours as the outline weight seems too large.
-Discussion, line 304-306: "… From these studies, it is clear that the LHb microcircuitry is quite complex and when engaged by aggression can have varying effects depending on the cell type and downstream projection. …" -I'd suggest to delete 'quite'.
Reviewer #3 (Remarks to the Author): Takahashi et al. have studied the circuit components behind how social provocation ("instigation") elevates intermale aggression. By combining tracing and opto-and chemogenetic manipulation, they show that LHb-DRN neurons contribute to this escalation effect of prior exposure to (but not direct contact with) another male, but that activity in this pathway does not appear to be related to the baseline levels of aggression (as examined in a resident-intruder paradigm). They identify non-5HT raphe neurons as key to this phenomenon, and further show that a projection from DRN to the VTA is the downstream mediator of instigation-induced escalation of intermale aggression. This is an overall well-designed and -executed study that provides an anatomical framework to the curious -and potentially clinically relevant -role of provocation in augmenting aggression. Several important controls are included (e.g. testing the phenomenon in two separate animal facilities, which I enjoyed seeing presented). There are some issues, esp with regards to with interpretation, that could improve the ms. Some control experiments also need to be added.

MAJOR ISSUES
1. Fig 2, chemogenetic inhibition: I failed to understand if control animals were also injected with CNO (i.e. animals not expressing hM4D)? I.e. is there an effect of CNO independent of the transgene? If not, this is a control that should be included. 2. Fig 4, optogenetic stimulation: the authors hardly discuss the lack of effect in the ON/OFF condition at all. But is this not the crucial experiment? i.e. the activity of the LHb-DRN projection in the period just prior to the introduction intruder is the neuronal correlate to instigation and thus would be expected to yield the same effect as the Inst condition -but it does not. This (negative) result seems as if it argues against the hypothesis. Please address this issue (also in the Results-Discussion) and clarify how it fits into the overall picture. 3. Fig 5b: please include quantification and statistics of the validation of neuronal silencing. 4. Line 123-125: Please clarify how many cells were counted from how many animals. The details on histochemical quantification are overall a bit too brief in this manuscript. This should also be clarified in Fig 4e, f,and in Fig 6g. 5. I am confused by part of the argument the authors are making for the proposed circuit. On p. 12, lines 326-7 it is described that that the DRN target neurons of the LHb projection are serotonergic or GABAergic (I am assuming that these are two separate populations?). But as I understand the model, the LHb and DRN neurons in the LHb-DRN-VTA chain should be excitatory, e.g. to explain why optogenetic stimulation has the effect shown in Fig 7. Can the authors please address this apparent paradox? What is the relationship between the GABAergic and the Vglut3 neurons in the DRN (referenced on line 333-4)?

MINOR ISSUES
6. The manuscript should emphasize for the reader that the authors are examining *intermale* aggression. I understand that for reasons of brevity this is generally shortened to just aggression, but the non-expert reader should be made aware that this is one of several forms of aggression observed in the mouse. 7. Line 55 (and in Discussion): it is not clear to me what justifies the implication of aggressive "arousal" in this context. Please elaborate. 8. p. 9, lines 224-234: why was ArchT used in these experiments and not NpHR3.0 8as in Fig 2)? Please explain rationale. 9. p. 12, lines 323-5 ("The mammalian LHb…"): the message/conclusion of this sentence was not entirely clear to me. 10. p. 14, lines 355-7 ("Thus, it is possible…"): The logic of how instigation should be equivalent to omission of an expected reward is not clear to me. Please elaborate on this argument. 11. p. 14, p. 361-362: ("Although a large body of evidence…") please add reference(s) for this sentence. 12. p. 14, pg starting on line 372: can the authors please explain how they envision the relationship between instigation and provocation in humans? Are these synonymous terms or is there a difference? If so, what is the difference? 13. Methods: the sources of reagents, equipment etc. is given in an inconsistent manner, sometimes with sometimes without geography, sometimes states are written out in full sometimes in acronyms etc. Please standardize. 14. p. 17  20. Some grammatical and spelling issues were noted: Line 196: "their" -I assume this refers to the animals but the sentence is not very well constructed. Line 232: "firings" I don't think this is a term that one would use. Consider e.g. "action potential discharge" or "firing rate". Line 279: "…light stimulation, was observed…" Remove comma. Line 285: I understand this is up for discussion nowadays but in this referee's book data is plural. Line 336: "has", please change to "have". Line 357: "rewards" should read "reward". Lines 428-30: "Cob" spelled with one "b" I think? Line 433: "Institute" should read "Institutes" Line 947: "tesst" should read "tests" Figures 2, 3, 5, 7: "Freqency" should read "Frequency". Ex data Fig 5: "fiver" should read "fiber"

Point-by-point response to the reviewers' comments
We would like to thank the reviewers for their helpful comments on our manuscript. Below is a detailed point-by-point response to the reviewer concerns. We have denoted our responses to the reviewer comments in red text. Changes have been highlighted in yellow throughout the manuscript.
Reviewer #1 (Remarks to the Author): The manuscript "Lateral habenula glutamatergic 1 neurons projecting to the dorsal raphe nucleus promote aggressive arousal" by Aki Takahashi  The authors conclude that the LHb glutamatergic projections to the DRN escalate aggressive behavior by activating non-serotonergic DRN neurons projecting to the VTA. This study identifies a DRN input and the DRN cell type that are involved in social instigation induced aggression escalation. These findings are interesting and improve understanding of the circuit mechanism for the regulation of aggressive behavior. However, this study is preliminary with some major concerns that need to be addressed.
We really appreciate the reviewer's positive and thoughtful comments that have significantly improved this manuscript.
Specific comments: 1. The authors cited their previous microdialysis study which showed that glutamate in the DRN is increased during aggressive behavior and social instigation for their claim that the LHb-DRN projections are activated by social instigation. This study did not specifically measure neural activities of the LHb-DRN projections. Although they used c-fos labeling to show that the DRN projection neurons of the LHb are activated specifically by social instigation, c-fos does not report the specific pattern of neural activities and has a poor temporal resolution. As the LHb-DRN projection is the main topic of this study, it is critical to confirm activation of the LHb-DRN projection neurons, the temporal relationship between LHb neuron activation, social instigation, and aggressive behavior, firing patterns, and the consequence of LHb neuron activation on DRN neuron activities with calcium imaging or electrophysiology.
We agree with the reviewer that c-Fos has low temporal resolution and it may not accurately reflect changes in neural firing patterns. As suggested by the reviewer, we initially attempted to record the activity of the LHb-DRN projection neurons using GCaMP, but due to technical difficulties we were unable to record accurate, reliable Ca 2+ signals within this pathway. As an alternative, we conducted ex vivo slice electrophysiology in LHb-DRN projecting neurons and found an increase in firing rate and an increase in resting membrane potential after social instigation-heightened aggression relative to mice that only experienced standard aggression in the resident intruder (RI) test. Our data also showed that social instigation-heightened aggression increased the number of LHb-DRN neurons exhibiting spontaneous activity. We believe that this new data complements and supports existing c-Fos expression data confirming that social instigation-heightened aggression activates the LHb-DRN neurons compared to standard aggression in the RI test. We have now added this data as a new Fig. 2. 2. The experiment of optogenetically activating the DRN-VTA projections does not support that the LHb increases aggression through the DRN-VTA projections as there is no evidence that this manipulation mimics the activation of LHb-DRN projections by social instigation. The conclusion based on this experiment that the LHb inputs to the DRN promote aggression by activating VTA projecting DRN neurons is not grounded.
We thank the reviewer for highlighting this important point. We performed an additional experiment to examine whether activation of the DRN-VTA projection mimics the effect of social instigation. To do this, we stimulated the DRN-VTA projection just prior to RI test to see whether this increases aggressive behavior. We found that ON/OFF stimulation of DRN-VTA projection, as well as ON and ON/ON stimulations, increased intermale aggressive behavior compared to the OFF session. Thus, activation of this pathway mimics the effect of social instigation, suggesting a possible involvement of DRN-VTA projections on instigation-heightened aggression.
We now show this data in a new Fig. 8. On the other hand, this is still indirect evidence to conclude that di-synaptic connections of LHb-DRN-VTA are involved in instigation-heightened aggression. Therefore, we mention this important limitation and temper our conclusions by suggesting that the VTA-projecting non-serotonergic DRN neurons may be one of the potential targets, but further work is needed to confirm the di-synaptic nature of this projection (see abstract, introduction, and discussion).
3. The rationale of studying LHb is unclear as DRN receives glutamatergic projections from many brain areas besides LHb, including the prefrontal cortex, hypothalamic areas, and the extended amygdala.
We did analyze c-Fos in several other upstream regions including the lateral hypothalamic area (LH)-DRN projection and did not find evidence that instigation-heightened aggression-activated these relative to a group receiving the standard RI test alone (see Extended Data Fig S2). While we did not analyze prefrontal cortex inputs to DRN, previous studies have shown that the mPFC sends a projection to DRN that promotes social avoidance behavior in the social defeat stress in the mouse (Challis et al 2014 Front Behav Neurosci). In the dominant hamster, the mPFC-DRN projection is activated by acute social defeat stress and this promotes a stress resilient phenotype (Gizzell et al 2020 Front Neural Circuits). Therefore, neural inputs from the mPFC to the DRN play an important role in social behavior, and we agree that this projection will need to be investigated in future studies to see if they also regulate aggressive behavior. We've included this in the discussion section on page 15. Supplemental Fig 1 and missing  We thank the reviewer for this suggestion. Attack latency is an important indice of aggressive behavior. We've now added new supplemental Figs (Extended Data Fig. 3, 4 , 5, 7, 8, 10, 11) to show the results of all behaviors analyzed including the attack latency. Please note that for some studies we conducted longer habituation sessions before starting optogenetics/chemogenetics experiments in order for animals to show stable aggressive behavior. Many of the animals in these studies with extended habituation showed very short attack latency even without social instigation. Therefore, we could not detect a significant effect of manipulations in this index in some of the experiments. 5. The image quality is generally poor. Region of interest (e.g. DRN) should be outlined. Highmagnification images should be provided for co-localization experiments in Fig 1f-h, Fig 2C. We have increased the image quality, and outlined the region of interest (DRN, LHb). Also, Fig1fh were substituted with magnified images and an enlarged picture was inserted in Fig. 3c (corresponds to the previous Fig 2c).

Attack latency is only shown in
6. The authors found that both the ON and ON/ON stimulation schemes increased aggressive behavior compared to OFF sessions, but there is no statistically significant difference between on/off and off (Fig. 4i,j). These results are contradictory to the claim that the LHb-DRN projection is responsible for aggression escalation induced by social instigation, which predicts that the ON/Off scheme should increase aggression. This is an important point also raised by Reviewer 3. We have now added an additional experiment to examine the effect of LHb-DRN ON/OFF stimulation in a new batch of animals.
We hypothesized that LHb-DRN activation would need to be combined with a social stimulus in order to elicit "aggressive arousal". Therefore, we conducted a short-term (1 min) social instigation test that we termed subthreshold social instigation, which alone was not enough to produce pro-aggressive effects. Our results show that a combination of subthreshold social instigation paired with optogenetic ON/OFF stimulation of LHb-DRN projection caused a significant increase in aggressive behavior compared to RI test alone or short-term instigation without optogenetic stimulation. We've added these new data to Fig. 5o-q, as well as to Extended Data Fig. 7. 7. There are some inappropriate statements: "In particular, continuous activation of the LHb-DRN projection prior to an aggressive encounter caused a strong escalation of aggressive behaviors". This is not a proper interpretation of Figure   4 as the ON/ON regime does not distinguish if stimulation before or during aggressive encounter elevates aggression.
We now exclude this sentence from the main text.
"Thus, our data show that depending on the subregion of the LHb or neural projection targets, activation of LHb neurons can have different effects on aggressive behavior." This manuscript does not have data on LHb subregions.
We agree with the reviewer and have modified the discussion accordingly.
8. In Figure 6G, eArchT-EYFP does not delimit the contour of a cell. How to use YFP signals to determine if mcherry cells are YFP positive?
As the reviewer points out, it is hard to identify EYFP+ cells by using eArchT-EFYP. However, it is still possible to observe the cell-body-like round shape surrounding DAPI, which labels the nuclei. Thus, we counted the EYFP+ cells when the edge of mCherry (or DAPI) were surrounded by EYFP. We also conducted an additional experiment by labeling serotonin neurons with Tph2 to assess its co-localization with EYFP (Extended Data Fig. 9a-e). Higher magnification images are now included in Fig. 7. 9. DRN neurons project to many brain regions besides VTA. Why focus on the VTA projections?
We thank the reviewer for highlighting this point. It was not clear in our original manuscript why we chose to focus on the VTA among other projection areas. Several previous studies have shown that VTA DA is implicated in escalated aggression and aggression reward (for review see . From these important studies, as well as our own data, which shows a substantial projection from the DRN to VTA, we chose the VTA as a first target to examine in this study. We've added this rationale to the newly revised manuscript on page 12 and also discuss the necessity of examining other projection targets on aggression in future studies.
10. In the experiment of optogenetic activation of the DRN-VTA projections, an on/off session needs to be added to test if activating these project¬¬ions mimics social instigation.
We appreciate the reviewer's advice here. As we mentioned in response to point # 2 above, we've 11. An off-off-off session should be added to Fig. 7 to test if the decrease over time of the duration of total aggressive behaviors (Fig. 7g) and locomotion (Fig. 7j) is due to habituation effects.
As mentioned in the previous critique, we've now added new data in Fig. 7 that shows no indication of a habituation effect in the new data set.

Reviewer #2 (Remarks to the Author):
Takahashi and colleagues investigated the role of lateral habenula (LHb) input to the dorsal raphe (DRN) in mediating aggression. Their results suggest that glutamatergic LHb input to the DRN plays a key role in escalating aggression, but not in basal aggressive behaviour. The authors went on to show that DRN neurons that receive LHb input project to the ventral tegmental area (VTA). Optogenetic activation of the latter projection (DRN-VTA) also increases aggressive behaviour.
Overall, the study is well designed and the topic is timely. The authors have used state-of-theart techniques and as far as I can see almost all important control experiments were performed to justify the conclusion mentioned above.
I am certain that this study is of interest to a wider community, and thus, the study deserves publication in this journal.

I only have a few minor comments:
We really appreciate the reviewer's positive and thoughtful comments that have significantly improved this manuscript. A 4-6 week incubation period is necessary for opsins to be expressed at high enough levels in the terminal for circuit specific stimulation protocols. We have added the following sentence in the method section to explain this: "Behavior tests with optical stimulation was started at least five weeks after AAV injection for optimal expression of the opsin in the projection terminal 33 ". By contrast, for DREADD experiments we typically wait 3-4 weeks because we observe strong expression of hM4D-EYFP within the soma at this time point. In fact, as we describe in the methods, the first CNO injection for these studies was administered 4 weeks after the AAV injection.
-In terms of the electrophysiological experiments you tested the Arch construct in vitro ( Figure   5b), but for the other paradigms you did not test the constructs and basically their efficacy (e.g. for the hM4D-, NpHR-and ChR2-experiments). For example, did the one-minute light stimulation you applied to activate LHb-DRN projections (Figure 4) Fig. 6). Also, circuit specific c-Fos assessment showed no difference in c-Fos expression or c-Fos colocalization with RetroBead between right and left LHb. We have now added a sentence in the main text to mention that there was no functional asymmetry in the RetroBeads experiment (Line 139-141).
-Extended Data Fig. 4e and j -It is difficult to recognise the legend colours as the outline weight seems too large.
We've modified the outline weight to have better visibility.
-Discussion, line 304-306: "… From these studies, it is clear that the LHb microcircuitry is quite complex and when engaged by aggression can have varying effects depending on the cell type and downstream projection. …" -I'd suggest to delete 'quite'.
We deleted "quite" from the sentence.
Reviewer #3 (Remarks to the Author): Takahashi et al. have studied the circuit components behind how social provocation ("instigation") elevates intermale aggression. By combining tracing and opto-and chemogenetic manipulation, they show that LHb-DRN neurons contribute to this escalation effect of prior exposure to (but not direct contact with) another male, but that activity in this pathway does not appear to be related to the baseline levels of aggression (as examined in a resident-intruder paradigm). They identify non-5HT raphe neurons as key to this phenomenon, and further show that a projection from DRN to the VTA is the downstream mediator of instigation-induced escalation of intermale aggression. This is an overall well-designed and -executed study that provides an anatomical framework to the curious -and potentially clinically relevant -role of provocation in augmenting aggression. Several important controls are included (e.g. testing the phenomenon in two separate animal facilities, which I enjoyed seeing presented). There are some issues, esp with regards to with interpretation, that could improve the ms. Some control experiments also need to be added.
We really appreciate the reviewer's positive and thoughtful comments that have significantly improved this manuscript.

MAJOR ISSUES
1. Fig 2, chemogenetic inhibition: I failed to understand if control animals were also injected with CNO (i.e. animals not expressing hM4D)? I.e. is there an effect of CNO independent of the transgene? If not, this is a control that should be included.
The control EYFP animals also received CNO at the same time as hM4D-expressing animals.
Because CNO injection (3 rd session (Inst) or 7 th session (RI)) did not elicit any significant differences in behavior compared to saline injection (2 nd & 5 th sessions (Inst) or 6 th session (RI)) in the control animal, we concluded that there was no effect of CNO on aggressive behaviors.
2. Fig 4, optogenetic stimulation: the authors hardly discuss the lack of effect in the ON/OFF condition at all. But is this not the crucial experiment? i.e. the activity of the LHb-DRN projection in the period just prior to the introduction intruder is the neuronal correlate to instigation and thus would be expected to yield the same effect as the Inst condition -but it does not. This (negative) result seems as if it argues against the hypothesis. Please address this issue (also in the Results-Discussion) and clarify how it fits into the overall picture.
We thank the reviewer for pointing this out. This is an important point also raised by Reviewer 1. Please see response #2 to Reviewer 1.
3. Fig 5b: please include quantification and statistics of the validation of neuronal silencing.
We have now conducted a new set of slice recordings to confirm the eArchT inhibition of serotonergic neurons (5 cells from 3 animals). We confirmed that the yellow light illumination significantly reduced spontaneous firing and reduced resting membrane potential. Also, we show that eArchT+ cells recovered spontaneous firing immediately after the 10 min of light illumination (Fig 6a-c). Because the recording protocol was modified for this new experiment (i.e. from loose cell attached recordings to whole cell patch-clamp recording), we've altered the method section accordingly. We apologize for our misleading description. In addition to serotonergic and GABAergic neurons, the DRN contains glutamatergic, dopaminergic, and peptidergic neurons. In this study, we show that a majority of DRN neurons that receive LHb input were not Tph2+ ( Fig. 7a- Therefore, these results suggest that LHb-DRN-VTA circuit is excitatory.

MINOR ISSUES
6. The manuscript should emphasize for the reader that the authors are examining *intermale* aggression. I understand that for reasons of brevity this is generally shortened to just aggression, but the non-expert reader should be made aware that this is one of several forms of aggression observed in the mouse.
We now state "intermale aggression" throughout the manuscript. We have also added new data where we examine whether activation of LHb-DRN projection triggers aggression towards females, but found no evidence for female-directed aggression (new Fig 5r). We thank the reviewer for bringing up this important point. We operationally defined the internal state that increases aggressive behavior by social instigation as "aggressive arousal", based on the definition originally proposed by Michael Potegal. "Aggressive arousal" has been considered as theoretically distinguishable from "general arousal" (proposed as behavior-specific drive by D.E. Berlyne), as it specifically affects aggressive components of behaviors but not other behaviors reflective of general arousal states, such as locomotor activity or sexual behavior. To examine the effect of activation of the LHb-DRN projection in greater detail, we examined whether stimulation affects locomotor activity or female-directed behavior. Our results show that LHb-DRN stimulation did not increase locomotor activity, nor did it induce sexual behavior towards a female (new Fig. 5r, Extended Data Fig. 7j). Therefore, activation of LHb-DRN projection specifically enhances intermale aggression without affecting other arousal-related behaviors. We provide a more sophisticated explanation of aggressive arousal in the introduction (Line 85-86). 8. p. 9, lines 224-234: why was ArchT used in these experiments and not NpHR3.0 8as in Fig 2)?
Please explain rationale.
Previous studies have shown that ArchT is a better opsin for long-term neural silencing than eNpHR3.0 (Chow et al 2010 Nature, Tsunematsu et al 2013 Behav Brain Res). Because this study aimed to suppress 5-TH neural activity for up to 10 min (5 min Inst + 5 min RI), we decided to use eArchT3.0. Indeed, our electrophysiological recording shows successful inhibition of 5-HT neurons for 10 min by eArchT3.0 illumination (Fig. 6). By contrast, for the optogenetic inhibition of projection terminals, it has shown that ArchT can have a paradoxical excitatory effect (Mahn et al 2016 Nat Neurosci) and NpHR3.0 is considered to be desirable for this purpose. Therefore, we used NpHR3.0 for LHb-DRN terminal inhibition experiments with previously published illumination protocols (Fig. 4). This is also now described in the method section. 9. p. 12, lines 323-5 ("The mammalian LHb…"): the message/conclusion of this sentence was not entirely clear to me.
We have modified the text to clarify our conclusion.
10. p. 14, lines 355-7 ("Thus, it is possible…"): The logic of how instigation should be equivalent to omission of an expected reward is not clear to me. Please elaborate on this argument.
Previous studies have shown that the expression of aggressive behavior is rewarding to male mice and in the social instigation test, the resident male's motivation to express aggressive behavior towards an intruding rival is hampered by the existence of the protective cage, which might be similar to an omission of the reward. It has been shown that omission of expected rewards (either food or water) causes escalation of aggressive behavior in the mouse and pigeon.
Although this is just speculation, we think this possibility will be interesting to examine in future studies. We have now included this elaborated discussion, however, we are also happy to remove it if the reviewer does not feel it is warranted. 11. p. 14, p. 361-362: ("Although a large body of evidence…") please add reference(s) for this sentence.
This has been added. 12. p. 14, pg starting on line 372: can the authors please explain how they envision the relationship between instigation and provocation in humans? Are these synonymous terms or is there a difference? If so, what is the difference?
While instigation and provocation are thought to tap into similar processes, there are some key differences that we now highlight in the manuscript. Commonly used methods for provocation in human studies within a laboratory setting involve the test subject receiving punishment (i.e. electrical shock or point subtraction) from a fictitious opponent in a competitive game. In the social instigation procedure, animals encounter an opponent via sensory contact but the test subject does not receive punishment per se. Both provocation and instigation involve a potentially hostile rival, and both increase aggressive behavior. We have modified the discussion to describe this potential relationship.
13. Methods: the sources of reagents, equipment etc. is given in an inconsistent manner, sometimes with sometimes without geography, sometimes states are written out in full sometimes in acronyms etc. Please standardize.
This has been corrected for consistency 14. p. 17, lines 436-8: why were the DRN-VTA ChR2 experiments conducted during the light cycle unlike other experiments? Please explain rationale in the Methods section.
As the reviewer pointed out, this experiment was conducted in the ISMMS facility where the light-dark cycle was not reversed. Because one reviewer asked to examine the effect of ON/OFF stimulation of DRN-VTA projection to determine whether this projection is capable of mimicking the effect of social instigation, we conducted a new experiment to examine the effect of ON, ON/OFF, and ON/ON stimulation schemes of DRN-VTA projection in the dark cycle in the animal facility of the University of Tsukuba. We found that activation of the DRN-VTA projection also increased intermale aggressive behavior in the dark cycle confirming our earlier studies that